PTC124 (Ataluren)

PTC124 is a selective inhibitor of nonsense mutations with IC50 value of 0.1μM [1].
Nonsense mutation is a point mutation in a sequence of DNA which promotes premature translational termination. Different from missense mutation, nonsense mutation means a single nucleotide is changed and results in the substitution of a different amino acid. It has been reported that some genetic disorders (thalassemia, DMD, CF, Hurler syndrome) are correlated with nonsense mutation [1, 2].
PTC124 is a potent nonsense mutations inhibitor. When tested with human or mdx mice primary muscle cells expressing dystrophin nonsense alleles, 2-8 weeks treatment of PTC124 enhanced the production of dystrophin [1]. In iPSC-derived RPE cells with nonsense mutation c.519C>T (p.R120X), PTC124 treatment restored endogenous, full-length RP2 protein with near 20% [3]. When tested with COS7 cells carrying the nonsense mutation pDsRed-EGFPmtag-Y445X, EGFP transcript level was increased after treated by PTC124 in a dose-dependent manner [2].
In mdx mice model, oral administration of PTC124 for 2-8 weeks rescued striated muscle function [1]. In a model of Cftr-/- mice expressing a human CFTR-G542X transgene, s.c. injection or oral administration of PTC124 restored a considerable amount of human (h) CFTR protein and function via suppressing G542X nonsense mutation [4].
References:
[1].    Welch, E.M., et al., PTC124 targets genetic disorders caused by nonsense mutations. Nature, 2007. 447(7140): p. 87-91.
[2].    Shen, Q., P. Guo, and B. Chai, pDsRed-EGFPmtag-, an effective dual fluorescent reporter system for cell-based screens of premature termination codon. Cytotechnology, 2014.
[3].    Schwarz, N., et al., Translational read-through of the RP2 Arg120stop mutation in patient iPSC-derived retinal pigment epithelium cells. Hum Mol Genet, 2015. 24(4): p. 972-86.
[4].    Du, M., et al., PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model. Proc Natl Acad Sci U S A, 2008. 105(6): p. 2064-9.

Toward a rationale for the PTC124 (Ataluren) promoted readthrough of premature stop codons: a computational approach and GFP-reporter cell-based assay.[Pubmed:24483936]

Mol Pharm. 2014 Mar 3;11(3):653-64.

The presence in the mRNA of premature stop codons (PTCs) results in protein truncation responsible for several inherited (genetic) diseases. A well-known example of these diseases is cystic fibrosis (CF), where approximately 10% (worldwide) of patients have nonsense mutations in the CF transmembrane regulator (CFTR) gene. PTC124 (3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)-benzoic acid), also known as Ataluren, is a small molecule that has been suggested to allow PTC readthrough even though its target has yet to be identified. In the lack of a general consensus about its mechanism of action, we experimentally tested the ability of PTC124 to promote the readthrough of premature termination codons by using a new reporter. The reporter vector was based on a plasmid harboring the H2B histone coding sequence fused in frame with the green fluorescent protein (GFP) cDNA, and a TGA stop codon was introduced in the H2B-GFP gene by site-directed mutagenesis. Additionally, an unprecedented computational study on the putative supramolecular interaction between PTC124 and an 11-codon (33-nucleotides) sequence corresponding to a CFTR mRNA fragment containing a central UGA nonsense mutation showed a specific interaction between PTC124 and the UGA codon. Altogether, the H2B-GFP-opal based assay and the molecular dynamics (MD) simulation support the hypothesis that PTC124 is able to promote the specific readthrough of internal TGA premature stop codons.

Enhancement of premature stop codon readthrough in the CFTR gene by Ataluren (PTC124) derivatives.[Pubmed:26142488]

Eur J Med Chem. 2015 Aug 28;101:236-44.

Premature stop codons are the result of nonsense mutations occurring within the coding sequence of a gene. These mutations lead to the synthesis of a truncated protein and are responsible for several genetic diseases. A potential pharmacological approach to treat these diseases is to promote the translational readthrough of premature stop codons by small molecules aiming to restore the full-length protein. The compound PTC124 (Ataluren) was reported to promote the readthrough of the premature UGA stop codon, although its activity was questioned. The potential interaction of PTC124 with mutated mRNA was recently suggested by molecular dynamics (MD) studies highlighting the importance of H-bonding and stacking pi-pi interactions. To improve the readthrough activity we changed the fluorine number and position in the PTC124 fluoroaryl moiety. The readthrough ability of these PTC124 derivatives was tested in human cells harboring reporter plasmids with premature stop codons in H2BGFP and FLuc genes as well as in cystic fibrosis (CF) IB3.1 cells with a nonsense mutation. Maintaining low toxicity, three of these molecules showed higher efficacy than PTC124 in the readthrough of the UGA premature stop codon and in recovering the expression of the CFTR protein in IB3.1 cells from cystic fibrosis patient. Molecular dynamics simulations performed with mutated CFTR mRNA fragments and active or inactive derivatives are in agreement with the suggested interaction of PTC124 with mRNA.

A lack of premature termination codon read-through efficacy of PTC124 (Ataluren) in a diverse array of reporter assays.[Pubmed:23824517]

PLoS Biol. 2013;11(6):e1001593.

The drug molecule PTC124 (Ataluren) has been described as a read-through agent, capable of suppressing premature termination codons (PTCs) and restoring functional protein production from genes disrupted by nonsense mutations. Following the discovery of PTC124 there was some controversy regarding its mechanism of action with two reports attributing its activity to an off-target effect on the Firefly luciferase (FLuc) reporter used in the development of the molecule. Despite questions remaining as to its mechanism of action, development of PTC124 continued into the clinic and it is being actively pursued as a potential nonsense mutation therapy. To thoroughly test the ability of PTC124 to read through nonsense mutations, we conducted a detailed assessment comparing the efficacy of PTC124 with the classical aminoglycoside antibiotic read-through agent geneticin (G418) across a diverse range of in vitro reporter assays. We can confirm the off-target FLuc activity of PTC124 but found that, while G418 exhibits varying activity in every read-through assay, there is no evidence of activity for PTC124.

Chronic ataluren (PTC124) treatment of nonsense mutation cystic fibrosis.[Pubmed:21233271]

Eur Respir J. 2011 Jul;38(1):59-69.

In a subset of patients with cystic fibrosis (CF), nonsense mutations (premature stop codons) disrupt production of full-length, functional CF transmembrane conductance regulator (CFTR). Ataluren (PTC124) allows ribosomal readthrough of premature stop codons in mRNA. We evaluated drug activity and safety in patients with nonsense mutation CF who took ataluren three times daily (morning, midday and evening) for 12 weeks at either a lower dose (4, 4 and 8 mg.kg(-1)) or higher dose (10, 10 and 20 mg.kg(-1)). The study enrolled 19 patients (10 males and nine females aged 19-57 yrs; dose: lower 12, higher seven) with a classic CF phenotype, at least one CFTR nonsense mutation allele, and an abnormal nasal total chloride transport. Both ataluren doses were similarly active, improving total chloride transport with a combined mean change of -5.4 mV (p<0.001), and on-treatment responses (at least -5 mV improvement) and hyperpolarisations (values more electrically negative than -5 mV) in 61% (p<0.001) and 56% (p = 0.002) of patients. CFTR function was greater with time and was accompanied by trends toward improvements in pulmonary function and CF-related coughing. Adverse clinical and laboratory findings were uncommon and usually mild. Chronic ataluren administration produced time-dependent improvements in CFTR activity and clinical parameters with generally good tolerability.